Pupovac Aleta, Geraghty Nicholas J, Watson Debbie, Sluyter Ronald
1] School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia [2] Illawarra Health and Medical Research Institute, Wollongong, New South Wales, Australia.
Immunol Cell Biol. 2015 Jan;93(1):77-85. doi: 10.1038/icb.2014.69. Epub 2014 Aug 26.
Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.
细胞外损伤相关分子模式三磷酸腺苷（ATP）激活P2X7受体，可诱导包括低亲和力IgE受体CD23在内的细胞表面分子从人白细胞上脱落。整合素和金属蛋白酶（ADAM）10介导P2X7诱导的多发性骨髓瘤RPMI 8226 B细胞表面CD23的脱落；然而，这一过程是否发生在原代B细胞中尚不清楚。本研究的目的是确定P2X7激活是否会诱导原代人B细胞和鼠B细胞表面CD23的快速脱落。流式细胞术和酶联免疫吸附测定结果显示，ATP处理人B细胞和鼠B细胞可诱导CD23快速脱落。用特异性P2X7拮抗剂AZ10606120处理细胞，几乎完全抑制了ATP诱导的人B细胞和鼠B细胞表面CD23的脱落。P2X7基因敲除小鼠的B细胞中，ATP诱导的CD23脱落也受到抑制。通过对脾细胞进行免疫印迹以及对ATP诱导的脾B细胞和T细胞摄取YO-PRO-1(2+)进行流式细胞术检测，证实P2X7基因敲除小鼠中不存在全长功能性P2X7。广谱金属蛋白酶拮抗剂BB-94和ADAM10拮抗剂GI254023X均抑制了P2X7诱导的人B细胞和鼠B细胞表面CD23的脱落。这些数据表明，P2X7激活可诱导原代人B细胞和鼠B细胞表面CD23快速脱落，且这一过程可能由ADAM10介导。
